Mutation of the conserved amino acid Arg 702 to Cys (R702C) in nonmuscle myosin heavy chain (NMHC) II-A in humans leads to abnormalities in the kidney, platelets, leukocytes, inner ear and eye and mutation of N93K results in defects in the platelets and leukocytes. Mutation of the homologous residue (R709C) in NMHC II-B in mice results in defects in the heart and brain. To better understand the mechanism leading to these defects, we expressed nonmuscle HMM II-B with the R709C mutation as well as a second homologous HMM with the N97K mutation, using the baculovirus expression system. We compared the actin-activated MgATPase activity and results from the in vitro actin gliding filament assay with values obtained for wild-type nonmuscle HMM II-B. Phosphorylated wild-type HMM II-B was activated by actin (Vmax = 0.17+0.04s-1) somewhat more than the N97K mutant (Vmax = 0.12+0.04s-1), but considerably more than the R709C mutant HMM (Vmax = 0.05+0.003s-1). The N97K mutant HMM moved actin filaments at a decreased rate (wild-type, 0.18+0.03 um/s vs 0.05+0.005 um/s), but the R709C mutant HMM could not translocate actin filaments at all, but did bind reversibly to actin filaments. In a separate set of experiments, we characterized baculovirus-expressed HMM for both non-inserted nonmuscle HMM II-C and HMM II-C with an 8-amino acid insert near the ATP binding region. Similar to the findings for smooth muscle HMM, inserted NMHC II-C HMM had a higher actin-activated MgATPase activity (Vmax = 0.85+0.07s-1 vs 0.29+0.04s-1); and translocated actin filaments more rapidly (0.08+0.003 um/s) than non-inserted nonmuscle HMM II-C (0.03+0.005 um/s). These values should not only help in characterization of the biological properties of the two myosins, but also help in understanding the phenotypes of the mutant mice we are generating.
